Since its introduction as an analytical technique in early 1970s, (Karasek, F. W. Anal. Chem., 1974, 46, 710A-717A) ion mobility spectrometry (IMS) has been increasingly applied to the characterization of gas-phase ions in a number of applications, including quality control in semiconductor manufacturing processes, (Carr, T. W. Thin Solid Films, 1977, 45, 115-122) environmental monitoring of air and water, (Eiceman, G. A.; Garcia-Gonzalez, L.; Wang, Y.-F.; Pittman, B. Talanta, 1992, 39, 459-467) detection of explosives, (Lawrence, A. H.; Neudorfl, P. Anal.Chem., 1977, 50, 152-155) and chemical warfare agents and toxins (Kientz, Ch. E. J.Chrom. A, 1998, 814, 1-23, Hill, H. H.; Siems, W. F.; Louis R. H. St.; McMinn, D. G. Anal.Chem., 1990, 62,1201A-1209A). IMS is based on spatial separation of gas-phase ion species due to differences in their mobilities through a buffer gas, analogous to capillary electrophoresis (CE) in the condensed phase.
Coupling of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) to IMS has provided an impetus for expanding the realm of IMS capabilities to proteomics and other system biology applications. (Wittmer, D.; Chen, Y. H.; Luckenbill, B. K.; Hill, H. H. Anal. Chem., 1994, 66, 2348-2355, Gillig, K. J.; Ruotolo, B.; Stone, E. G.; Russell, D. H.; Fuhrer, K.; Conin, M.; Schultz, A. J. Anal. Chem., 2000, 72, 3965-3971) The enormous complexity of biological systems, (e.g., >20,000 different proteins may be expressed at detectable levels by a mammalian system (Aebersold, R.; Mann, M. Nature, 2003, 422, 198)) has challenged the separation and analysis power of existing approaches, and to this point has been most effectively addressed by combinations of orthogonal fractionation and separation techniques combined with mass spectrometry (MS) as a final separation stage due to its high sensitivity, resolution, broad dynamic range and accurate mass measurement capability. Protein detection and identification in a variety of important biomedical applications, including discovery of candidate biomarkers in human blood plasma for early cancer detection, represents a significant analytical challenge for condensed phase multidimensional separations coupled to MS, as many proteins of interest are expected at abundance levels far below that of higher abundance proteins. (Anderson, N. L.; Anderson, N. G. Mol. Cell. Proteomics, 2002. 1, 845-867) The large dynamic range of interest (>1010), coupled with issues that derive from biological variation, has greatly hindered proteomic approaches for effectively discovering low level candidate biomarkers in such biological fluids. Liquid chromatography (LC)-MS based proteome analysis of human blood plasma has generally involved the coupling of a high abundance protein depletion step with intensive protein and/or peptide level fractionation/separation techniques to obtain a greater analytical “depth of coverage”. This approach effectively transforms each sample into many samples, and thus reduces the number of individual samples that can be analyzed. At present, for example, it is not practical to perform in-depth proteomic studies involving several hundred individual human blood plasma samples. This throughput versus proteome analysis coverage tradeoff can be addressed by either increasing the depth of coverage in a single analysis or the throughput of current approaches. Since gas-phase ion separations are typically two to three orders of magnitude faster (˜10-100 ms) than fast reversed-phase (RP) LC separations of comparable separation power (˜5-10 min), IMS represents an attractive complementary orthogonal separation approach. When introduced between the condensed phase separation and MS analysis, IMS can potentially increase the total effective peak capacity of a fast RPLC-MS platform by over an order of magnitude without affecting the overall analysis speed, and thus help in addressing both the depth of coverage and throughput needs.
IMS, in turn, benefits from coupling with fast MS detection capable of acquiring the entire mass spectrum in a single scan. Young et al. first coupled a lower-pressure IMS to an orthogonal time-of-flight (TOF) analyzer to measure the formation and decomposition rates of hydrates of hydronium ion. (Young, C. E.; Edelson, D.; Falconer, W. E. J. Chem. Phys., 1970, 53, 4295-4302) More recently, this approach was used in combination with ESI for characterization of different biochemical compounds. (Guevremont, R.; Siu, K. W. M.; Wang, J.; Ding, L. Anal. Chem., 1997, 69, 3959-3965, Hoaglund, C. S.; Valentine, S. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem., 1998, 70, 2236-2242) Despite its attractiveness for higher throughput proteomic studies, the application of IMS-MS has been limited by low sensitivity primarily arising from ion losses at the IMS-MS interface and low duty cycle. Tang et al. have recently reported on ion lossless IMS-MS separations with an IMS drift tube incorporated between two electrodynamic ion funnels. (Tang, K.; Shvartsburg, A. A.; Lee, H.-N.; Prior, D. C.; Buschbach, M. A.; Li, F.; Tolmachev, A. V.; Anderson, G. A.; Smith, R. D. Anal. Chem., 2005, 77, 3330-3339) In that experiment, ions were trapped in an “hourglass” ion funnel for 50 to 100 ms at an elevated pressure of 4 Torr and then gated into the IMS drift tube in short 50 μs pulses. At the exit of the IMS drift tube, ion packets, spatially dispersed mainly due to thermal diffusion, were captured by a regular ion funnel followed by a short collisional quadrupole. (Spangler, C. E.; Colins, C. L. Anal. Chem., 1975, 47, 403-407) These developments have also recently been adopted by Clemmer and coworkers. (Koeniger, S. L., Merenbloom, S. I., Valentine, S. J, Jarrold, M. F., Udseth H. R., Smith, R. D., Clemmer, D. E. Anal. Chem., 2006, 78, 4161-4174) Though ion transmission drastically improved as compared to earlier implementations of IMS-oTOF design, lower efficiency of ion trapping/accumulation in the ion funnel still limits the ion utilization efficiency or duty cycle. (Hoaglund-Hyzer, C. S.; Clemmer, D. E. Anal. Chem., 2001, 73, 177-184).
The duty cycle of IMS with a continuous ion source can be improved using a Fourier transform (FT) approach. (Knorr, F. J.; Eatherton, R. L.; Siems, W. F.; Hill, H. H., Jr. Anal. Chem., 1985, 57, 402-406; St. Louis, R. H.; Siems, W. F.; Hill, H. H. Anal. Chem., 1992, 64, 171-177) Using two gates at the entrance and at the exit of the drift region allows the “front” and “exit” gate opening and closing voltages to be correlated with the drift time for ions of interest. This approach provided a 3- to 5-fold increase in the signal-to-noise ratio (SNR) for the ions of a specific drift time at any given moment. Obtaining improved sensitivity for all species in a single IMS separation, requires a multiplexing technique, such as the Hadamard transform (HT). HT has been extensively used in optical spectrometry for over five decades. (Harwit, M.; Sloane, N. J. Hadamard Transform Optics; Academic Press: New York, 1979) The concept of measuring different bundles of objects by weighing them in groups rather than individually was first proposed by Fellgett, and the resulting increase in accuracy is sometimes called the Felgett or multiplex advantage. (Felgett, P. The theory of infrared sensitivities and its application to investigations of stellar radiation in the near infrared, PhD thesis, Cambridge University; Fellgett, P. J. de Physique, 1967, 28, 165-171) If spectral line intensities are simultaneously detected in N measurements, the theoretical increase in SNR over a single measurement is then expected to be ˜√{square root over (N)}. Decker has experimentally demonstrated such an SNR gain by comparing the mercury vapor emission spectra obtained with both a monochromator and a Hadamard transform spectrometer. (Decker, J. A. Appl. Opt., 1971, 10, 510-514) HT has been successfully applied to TOF MS and capillary electrophoresis (CE), yielding an increase in the duty cycle up to 50%. In CE experiments with fluorescence detection, Kaneta et al have experimentally demonstrated an SNR increase by a factor of 8, which was in excellent agreement with the theoretically predicated value of 8.02. (Kaneta, T.; Yamaguchi, Y.; Imasaka, T. Anal.Chem., 1999, 71, 5444-5446) CE multiplexing was achieved by photodegradation of a light-absorbing analyte, an approach that would be difficult to implement with a complex biological sample. For an HT on-axis TOF MS a proof-of-principle has been demonstrated using both direct infusion and CE, although no comparison with conventional signal averaging was reported. (Brock, A.; Rodriguez, N.; Zare, R. N. Anal. Chem., 1998, 70, 3735-3741; Fernandez, F. M.; Vadillo, J. M.; Kimmel, J. R.; Weterhall, M.; Markides, K.; Rodriguez, N.; Zare, R. N. Anal. Chem., 2002, 74, 1611-1617). A 5- to 6-fold increase in sensitivity has recently been reported in HT measurements using atmospheric-pressure IMS separations (without MS). (Clowers, B. H.; Siems, W. F.; Hill, H. H.; Massick, S. M. Anal.Chem., 2006, 78, 44-51; Szumlas, A. W.; Ray, S. J.; Hieftje, G. M. Anal. Chem., 2006, 78, 474-4471) This improvement, however, falls short of the theoretically expected gain of 15 to 45 (the theoretical gain for a 13-bit sequence is
                                                        2              13                        -            1                          2            ≅      45.25        )    .The discrepancy can be explained, in part, by the fact that the encoding pseudo-random binary sequence used in these multiplexing experiments exceeded the ion mobility drift times by almost two orders of magnitude. As a result, only a small fraction of the sequence contributed to any SNR enhancement as compared to that of a conventional averaging approach.
In sum, a major potential attraction of the IMS-TOF MS platform is separation speeds exceeding that of conventional condensed phase separations by orders of magnitude. Known limitations of the IMS-TOF MS platforms that presently mitigate this attraction include the need for extensive signal averaging due to factors that include significant ion losses in the IMS-TOF interface and an ion utilization efficiency of less than ˜1% with continuous ion sources (e.g., ESI). Accordingly, there exists a need for improved methods of analyzing ions using an Ion Mobility Spectrometer, and there exists a further need for improved methods of analyzing ions in arrangements where an Ion Mobility Spectrometer is interfaced with a Time of Flight Mass Spectrometer.